Antibiotic complex from micromonospora arborensis

ABSTRACT

Micromonospora arborensis produces a plurality of antibiotics named herein antibiotic 68-1147 complex, when cultivated under controlled aerobic conditions. Two of the antibiotics, antibiotic 68-1147 I and antibiotic 68-1147 II are described and shown to be effective antibacterial agents, especially against gram positive bacteria.

This application is a continuation-in-part application of our copending application, Ser. No. 516,242, filed Oct. 18, 1974 (now abandoned).

This invention relates to a new antibiotic complex elaborated by a novel species of the genus Micromonospora. Mor particularly, this invention relates to antibiotic 68-1147 complex and to the microorganism which produces the same, namely, Micromonospora arborensis.

THE MICROORGANISM

Micromonospora arborensis sometimes referred to as M. arborensis was isolated from a soil sample collected in Ann Arbor, Michigan and on the basis of its taxonomical and physiological properties has been determined to be a new species of Micromonospora.

A viable subculture of Micromonospora arborensis was deposited and can be obtained from the permanent collection of the Northern utilization and Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois where it was given the ascension No. NRRL 8041.

In addition to the description set forth in the tables below, a further distinguishing characteristic of M. arborensis is its ability to elaborate the antibiotic 68-1147 complex. This invention embraces Micromonospora arborensis NRRL 8041 and mutants and variants thereof having the distinguishing characteristics of M. arborensis NRRL 8041.

Thus, in one of its aspects this invention is directed to producing the antibiotic 68-1147 complex which comprises cultivating Micromonospora arborensis or a mutant or variant thereof having the identifying characteristics of NRRL 8041 in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium and isolating antibiotic 68-1147 complex therefrom.

In another of its aspects, this invention relates to the use of antibiotic 68-1147 complex or components thereof as antibacterial agents.

The taxonomical morphological and physiological characeristics set forth hereinbelow are derived from art recognized techniques utilizing standard media. In the description of the microorganism, two color designators are employed. The first is a color name taken from the "Descriptive Color Name Dictionary" by Taylor, Knoche and Granville published by the Container Corporation of America (1950) U.S.A., with a color chip number corresponding to the color name, the chip number being taken from "The Color Harmony Manual," 4th Edition, 1958, also published by the Container Corporation of America. The second designator consists of a color name and number which refers to the synonym and near synonym found in the National Bureau of Standards, Circular 553, November 1, 1965 (U.S.A.).

                  TABLE I                                                          ______________________________________                                         Medium:                                                                        Yeast extract 0.1%, Dextrose 1.0%, CaCO.sub.3 0.1%, Agar 1.5%                           Observations                                                          Organism     Macroscopic    Microscopic                                        ______________________________________                                         Micromonospora                                                                            Growth moderate,                                                                              Spores spherical to                                  arborensis plicate, no aerial                                                                            ovate, 0.8 microns                                              mycelium.      average diameter.                                               g4Pc dusty orange,                                                             Moderate orange 53.                                                 ______________________________________                                    

                  TABLE II                                                         ______________________________________                                         GROWTH CHARACTERISTICS                                                         ______________________________________                                         Czapeks     Growth fair to poor, flat,                                         Medium      no aerial mycelium,                                                (Glucose)   no diffusible pigment.                                                         g3gc light tan, light yellowish brown 76.                          Asparagine- Growth fair, granular,                                             Glucose     no aerial mycelium,                                                Medium      faint yellow diffusible pigment produced.                                      g4nc russet orange, strong orange 50.                              Calcium     Growth fair to poor, flat,                                         Malate      no aerial mycelium,                                                Agar        no diffusible pigment.                                                         g3ic light amber, dark orange yellow 72.                           Nitrate     Variable.                                                          Reduction                                                                      Ordinary Agar                                                                              Growth poor, not recordable.                                       (water agar)                                                                   Nutrient    Growth fair, to poor, flat, furrowed,                              Agar        no aerial mycelium, very faint                                                 yellow diffusible pigment produced.                                            g3ic light amber, dark orange yellow 72.                           Loffler's   Growth fair, weakly plicate, no                                    Serum Medium                                                                               aerial mycelium, no diffusible pigment,                            (Difco)     substrate little decomposed.                                                   g41c dusty orange, moderate orange 53.                             Potato      Growth fair to poor.                                               Plug                                                                           Peptone     Growth poor, rarely one or two isolated                            Glucose     colonies developing, colonies,                                     Agar        when present, plicate no aerial mycelium,                                      diffusible pigment not apparent.                                               g4nc russet orange, strong orange 50.                              Egg Agar    Growth poor, substrate not decomposed.                             (Dorset Egg                                                                    Medium -                                                                       Difco)                                                                         Gelatin     Growth poor, flat, no aerial                                       Medium      mycelium, gelatin hydrolysis weak.                                 Starch      Growth fair, flat, furrowed,                                       Agar        no aerial mycelium, no diffusible                                              pigments, starch hydrolyzed.                                                   g4pe orange rust, deep orange 51.                                  Tyrosine    Growth poor, granular, no aerial                                   Medium      mycelium, dark amber (reddish brown)                                           diffusible pigment produced.                                                   Reaction positive.                                                 Litmus Milk Milk poorly peptonized.                                            (Difco)                                                                        Cellulose   Cellulose not decomposed.                                          Medium                                                                         Milk Agar   Growth moderate, plicate, no aerial                                            mycelium, yellow diffusible pigment                                            produced, milk hydrolyzed.                                                     g4nc russet orange, strong orange 50.                              Sucrose     Utilized                                                           Starch      Hydrolysis positive.                                               Temperature Grows at 26 and 37° C but not                                           at 50° C or above.                                          Bennett's   Growth fair, moderately plicate,                                   Agar        no aerial mycelium, light yellowish                                            diffusible pigment produced.                                                   g4pc russet orange, strong orange 50.                              Emerson's   Growth fair to poor, flat, no aerial                               Agar        mycelium, light yellowish                                                      diffusible pigment produced.                                                   g4nc russet orange, strong orange 50.                              Tomato Paste                                                                               Growth moderate, plicate to furrowed,                              Oatmeal Agar                                                                               no diffusible pigment apparent.                                                Periphery: g4nc russet orange, strong                                          orange 55. Center: g4ni brown spice, - moderate brown 58.          Tyrosine    Growth poor flat, no aerial mycelium,                              Agar        dark reddish brown diffusible pigment                                          abundantly produced. Reaction positive.                            Observations at                                                                2, 7, and 14 days                                                              (After Gordon and                                                              Smith J. Bact.                                                                 69:147)                                                                        Peptone     Growth fair, plicate-membranous,                                   Iron Agar   no aerial mycelium, yellow diffusible                              Observations at                                                                            pigment produced, no reaction.                                     2, 7, and 14 days                                                                          g3ic light amber, dark orange yellow 72.                           ______________________________________                                    

                  TABLE III                                                        ______________________________________                                         CARBOHYDRATE UTILIZATION                                                                           Growth                                                     ______________________________________                                         Negative                                                                       Control               Poor                                                     D-Arabinose           poor                                                     L-Arabinose           moderate                                                 Dulcitol              poor                                                     D-Galactose           moderate                                                 D-Glucose             moderate                                                 Glycerol              poor                                                     I-Inositol            poor                                                     B-Lactose             fair to poor                                             D-Levulose            moderate                                                 D-Mannitol            poor                                                     Mannose               moderate                                                 Melibiose             fair to poor                                             Melizitose            poor                                                     Raffinose             poor                                                     L-Rhamnose            poor                                                     D-Ribose              poor                                                     Salicin               poor                                                     Sucrose               moderate                                                 D-Xylose              moderate                                                 Cellulose             fair to poor                                             Starch                moderate                                                 Sorbitol              poor                                                     Control                                                                        0.5% yeast            poor                                                     extract                                                                        ______________________________________                                    

                  TABLE IV                                                         ______________________________________                                         UTILIZATION OF NITROGEN SOURCES                                                Nitrogen Source                                                                +1% glucose                                                                    ______________________________________                                         0.5% Difco Growth good, plicate, no aerial mycelium,                           Yeast Extract                                                                             faint yellowish diffusible pigment.                                            g5pa bright orange, vivid orange 48.                                1.0% NZ Amine                                                                             Growth moderate, plicate to membraneous,                            Type A     no aerial mycelium, faint                                                      yellowish diffusible pigment.                                                  g5nc burnt orange, strong reddish orange 35.                        1% Asparagine                                                                             Growth poor, not recordable.                                        1% Glutamic                                                                               Growth poor not recordable.                                         Acid                                                                           1% Sodium  Growth poor, not recordable.                                        Nitrate                                                                        1% Ammonium                                                                               Growth poor, not recordable.                                        Nitrate                                                                        ______________________________________                                    

Antibiotic 68-1147 is produced when the elaborating microorganism, M. arborensis is grown in an aqueous nutrient medium under submerged aerobic conditions. For small amounts of antibiotic, e.g. microgram to milligram quantities, surface culture in bottles or shake flasks may be employed. Typically, the nutrient medium is liquid, contains a carbon source such as an assimilable carbohydrate and an assimilable nitrogen source, such as a proteinaceous material. Preferred carbon sources include glucose, mannitol, levulose, sucrose, starch, ribose and the like. Preferred nitrogen sources include corn steep liquor, yeast extract soybean meal, meat peptones, casein hydrolysate, beef extract and the like. It is advantageous to use combinations of these carbon and nitrogen sources to provide good growth and antibiotic production.

Production of Antibiotic 68-1147 may be effected at most temperatures conducive to satisfactory growth of the microorganism; e.g. between 20° and 40° C, preferably 28°-35° C. Ordinarily, optimum production is obtained in 3-5 days in shake flasks and from about 2 to about 7 days in larger vessels. The pH of the medium is maintained between 6.0 and 8.5 during the fermentation, a pH of between 7 to 8 being preferred. Prior to sterilization and inoculation, the fermentation medium is usually adjusted to pH 7.0, re-adjustments being made as required during the course of the fermentation. Alternatively, the pH may be maintained at about 7.0 by the use of buffering agents such as calcium carbonate. These buffering agents generally tend to persist throughout the course of the fermentation.

When growth is carried out in large vessels and tanks, it is desirable to produce a vegetative inoculum of about 5% of the volume of the tank by inoculating the broth culture with a slant culture or a lyophilized culture of the organism. The inoculum stage of the fermentation usually requires from about 24 to about 120 hours with about 1 to 2 days being preferred. When a vigorously growing inoculum has been obtained, it is transferred aseptically to the larger vessel or tank containing sterile medium. During the course of the inoculum stage and the fermentation stage which follows, the tendency of the medium to foam is controlled by the addition of a suitable anti-foaming agent, such as, Dow Corning B®, The Dow Chemical Company, Midland, Michigan.

The medium in which the vegetative inoculum is produced may be identical to that utilized for the production of antibiotic or it may differ so long as the medium is one in which good growth of the microorganism is obtained.

Variations in the ratio of components of the antibiotic 68-1147 complex are observed when changes are made in the fermentation conditions. In fact, one component which is normally produced in inconsequential amounts is abundantly produced when the medium designated C below is employed as the fermentation medium. Thus, it is advisable to monitor the course of the fermentation by chromatography and bioautography in addition to performing assays at intervals.

The following media are useful for the production of the Antibiotic 68-1147 complex. Media A, B and C are especially suitable for antibiotic production whereas medium D is particularly useful for the preparation of the vegetative inoculum.

    ______________________________________                                         A     Yeast extract (General Biochemicals)                                                                   10 gm                                                  Mor-rex (Corn Products) 30 gm                                                  Calcium carbonate       4 gm                                                   Cobalt chloride         130 μg                                              Tap water               1000 ml                                          B     South African Fish solubles                                                                            10 gm                                                  Mor-rex                 30 gm                                                  Calcium carbonate       4 gm                                                   Cobalt chloride         130 μg                                              Tap water               1000 ml                                          C     Yeast extract (Difco)   5 gm                                                   N-Z amine (Sheffield Chemical)                                                                         5 gm                                                   Mor-rex                 30 gm                                                  Calcium carbonate       4 gm                                                   Cobalt chloride         130 μg                                              Tap water               1000 ml                                          D     Beef extract            3 gm                                                   Tryptose                5 gm                                                   Yeast extract           5 gm                                                   Dextrose                1 gm                                                   Starch                  24 gm                                                  Calcium carbonate       2 gm                                                   Tap water               1000 ml                                          ______________________________________                                    

The foregoing media are exemplary of the nutrients utilized by M. arborensis to produce antibiotic 68-1147 complex. However, it is obvious to those skilled in the fermentation art that a wide range of nutrients obtained from a number of suppliers may be substituted for the foregoing, and that generally good growth and antibiotic production can be obtained, such nutrients being the functional equivalent of those set forth herein.

Peak antibiotic production is determined on the basis of a cylinder cup assay using Staphyloccus aureus ATCC 6538P as the test organism. The physical conditions for the assay and the assay itself are substantially similar to that used for determining the antibiotic potency of samples containing penicillin. A suitable reference standard is one wherein 1 μg of material gives a zone of inhibition of 14.9 ±1.2 mm and may be assigned a potency of 1000 μg/mg.

When peak production is attained, the antibiotic complex may be isolated by methods known in the art. Extraction with a non-water miscible polar organic solvent is a preferred method for isolating the antibiotic complex. Extraction of the antibiotic complex is usually followed by concentration of the extracts and precipitation of the antibiotic by the addition of a non-polar organic solvent such as low molecular weight hydrocarbons.

Comparison of the antibiotic complex with other known antibiotics having substantial activity against Gram positive organisms via paper and/or thin layer chromatography and subsequent bioautography indicates the complex to be novel. The compounds with which the comparison was conducted included macrolides, penicillins, cephaosporins, actinomycins etc.

Antibiotic 68-1147 Complex

The antibiotic complex consists of a plurality of active components, however, only two have been produced in sufficient quantity for isolation and characterization. These two have been designated antibiotic 68-1147 I and antibiotic 68-1147 II the latter being a chromatographically slower moving and presumably a more polar component. In most, although not all, fermentations the component i.e. 68-1147 I is produced in greater abundance. Both component appear to contain the following amino acids: threonine, proline, alanine, cysteine, isoleucine and tyrosine.

                  TABLE V                                                          ______________________________________                                         Physicochemical Properties of Antibiotics 68-1147 I and II                     68-1147 I          68-1147 II                                                  ______________________________________                                         M.P. 229°-231° C dec.                                                             M.P. ≅ 300° C dec.                            ##STR1##                                                                                        ##STR2##                                                     [α].sub.D.sup.26° = -87.5°                                                  [α].sub.D.sup.26° = -65.5°                (C, 0.4%, dioxane)                                                                              (C, 0.4%, dioxane)                                            Elemental analysis                                                                              Elemental analysis                                            C, 49.46, H, 5.20; N, 14.88;                                                                    C, 50.24; H, 4.98; N, 15.87;                                  0, 16.18; S, 10.47                                                                              0, 16.24; S, 12.67 by difference                              The antibiotic has:                                                            an Infrared spectrum in                                                        mineral oil (Nujol) having                                                     characteristic absorption at:                                                  3.05 m,sp                                                                                9.00 w,sp                                                            3.45 s,sp                                                                                9.15 w,br                                                            5.78 w,sp                                                                                9.50 w,br                                                            6.08 s    9.40 w,br                                                            6.31 w,sp                                                                                9.75 w,sp                                                            6.60 s    9.95 sh                                                              6.65 s   10.58 w,br                                                            6.75 s   10.70 w,br                                                            7.30 m,sp                                                                               11.20 w,br                                                            7.45 sh  11.30 sh                                                              7.65 w,sp                                                                               11.90 microns w,br                                                    8.30 w,sp                                                                      ______________________________________                                          s = strong, m = medium, w = weak, sp = sharp, br = broad, sh = shoulder? 

Biological Properties of Antibiotic 68-1147 I and II

In Tables VI and VII are set forth the in vitro antibacterial spectrum of antibiotic 68-1147 I against a variety of microorganisms, including numerous resistant strains and against representative "anaerobes". The tests were performed via conventional tube dilution methods in Mueller-Hinton Broth.

Table VIII sets forth in vivo activity of antibiotic 68-1147 I in mice via subcutaneous and/or oral administration.

Table IX sets forth the in vitro antibacterial spctrum of antibiotic 68-1147 II against a variety of microorganisms.

                  TABLE VI                                                         ______________________________________                                         In Vitro Antibacterial Activity of Antibiotics                                 68-1147 I in Mueller-Hinton Broth                                                              MIC                                                            Organism        (mcg/ml) Resistant To*                                         ______________________________________                                         Staphylococcus                                                                             209P    0.03     --                                                aureus      Gray    0.03     --                                                            12      0.03     --                                                            Wood    0.03     P, L, T, S                                                    Zeigler 0.03     P, L, T, S                                                    512     0.08     P, L, T, S                                                    433     0.03     P                                                             St. M.  0.003    P, L, Ceph, S                                                 405     0.08     P, L, Ceph, S, T                                              237     0.3      P, L, Ceph, S, T                                              1613    0.08     P, L, Ceph, S, T                                              428     0.08     P, L, Ceph, S, T                                              1650N   0.03     P, L, Ceph, S, T                                              618     0.03     P, L, Ceph, S, T, E, 01                                       484     0.08     P, L, Ceph, S, T, E, 01                                       616     0.03     P, L, Ceph, S, T, E, 01                                       336     0.03     P, L, Ceph, S, T, E, 01                                       494     0.03     P, L, Ceph, S, T, E, 01                                       1141    0.03     P, L, Ceph, S, T, E, 01                                       1026    0.03     P, L, Ceph, S, T, E, 01                                       1179    0.03     P, L, Ceph, S, T, E, 01                                       468     0.03     P, L, Ceph, S, T, E, 01                                       979     0.03     P, L, Ceph, S, T, E, 01                                       282     0.03     P, L, Ceph, S, T, E, 01                                       618     0.03     P, L, Ceph, S, T, E, 01                           Enterococcus                                                                               685     0.03     P, Ceph, L, T, S                                              999     0.03     P, Ceph, L, T, S                                  Streptococcus                                                                              C       0.03                                                       pyogenes    27      0.03                                                                   30      0.03                                                                   16295   0.03                                                                   6589    0.03                                                                   3045    0.03                                                                   5       0.03                                                                   3       0.03                                                                   6       0.03                                                       Bacillus subtilis                                                                          0.03                                                               6633                                                                           E. Coli Sc  >25                                                                Klebsiella DA20                                                                            >25                                                                Proteus sp 12453                                                                           >25                                                                Pseudomonas >25                                                                aeruginosa Sc 8709                                                             Salmonella  >25                                                                schottmuelleri                                                                 ______________________________________                                          *P = penicillin, L = lincomycin, T = tetracycline, S = sulfonamide, Ceph       cephalothin, E = erythromycin, 01 = oleandomycin                         

                  TABLE VII                                                        ______________________________________                                         In Vitro Activity of Antibiotic 68-1147 I Against Anaerobes                    Organism                 MIC (mcg/ml)                                          ______________________________________                                         Corynebacterium acnes                                                          6923                     0.8                                                   6922                     3.0                                                   6912                     0.8                                                   6921                     0.3                                                   11827                    0.3                                                   Bacteroides fragilis     0.8                                                   Bacteroides melaninogenicus                                                                             37.5                                                  Bacteroides corrodens    3.0                                                   Clostridium septicium    0.03                                                  Clostridium novyi        0.03                                                  Peptostreptococcus sp.   0.01                                                  ______________________________________                                    

                  TABLE VIII                                                       ______________________________________                                         In Vivo Activity of Antibiotic 68-1147 I                                       Infecting Organism Route   PD.sub.50 (mg/kg)                                   ______________________________________                                         Staphylococcus aureus                                                           Gray              S. C.   0.2                                                                    Oral    >50                                                                    Acute Toxicity                                                               Route LD.sub.50 (mg/kg)                                                        S. C. >800                                                    ______________________________________                                    

                  TABLE IX                                                         ______________________________________                                         In Vitro Antibacterial Activity of Antibiotic                                  68-1147 II in Mueller-Hinton Broth                                                                     MIC                                                    Organism                (mcg/ml)                                               ______________________________________                                         Staphylococcus aureus                                                                           209P       .005                                                                Wood       .005                                                                Ziegler    .005                                                                59N        .005                                               Streptococcus pyogenes                                                                          C          .005                                                                27         .005                                               Bacillus subtilis 6623      .005                                               Pseudomonas aeruginosa                                                                          NRRL 3223  >25                                                ______________________________________                                    

Antibiotic 68-1147 complex and the components thereof are active against bacteria especially against gram-positive bacteria. The compounds are also active against certain species of anerobic bacteria. Thus, they may be used in combination with soaps and detergents to destroy or inhibit bacteria in hospitals especially in hospital lavatories and operating rooms. Additionally the antibiotics may be incorporated into formulations used for cleaning surgical instruments and the like. The antibiotic complex may also be used to treat infected laboratory animals to prevent the spread of infection.

EXAMPLE I Production of Antibiotic 68-1147 Complex

A. inoculum Preparation

Prepare a series of 300 ml shake flasks containing 70 ml of the following medium:

    ______________________________________                                         Beef Extract       3        g.                                                 Tryptose           5        g.                                                 Yeast Extract      5        g.                                                 Dextrose           1        g.                                                 Starch             24       g.                                                 Calcium Carbonate  2        g.                                                 Tap water          1000     ml.                                                ______________________________________                                    

Adjust to pH 7.5 then sterilize the medium at 121° C (15 psig) for 20 minutes, cool to room temperature (20°-25° C. Add a loopful of Micromonospora arboresnis from an agar slant or 0.5 ml of whole broth from a previously prepared inoculum. Incubate with continual agitation at 250-300 rpm for 2 days at 28° C.

b. fermentation

Prepare, sterilize, cool and adjust to pH 7.0 10 liters of the following medium in a 14 liter fermentor.

    ______________________________________                                         Yeast Extract        100      g.                                               Mor-rex (Corn Products)                                                                             300      g.                                               Calcium Carbonate    40       g.                                               Cobalt chloride      1.3      g.                                               Tap water            10       liters                                           ______________________________________                                    

Inoculate the medium with 500 ml of inoculum, prepared as described in step A. Incubate the fermentation mixture at 28° C for from about 66 to about 90 hours, until peak antibiotic production is attained as determined by standard cylinder cup assay against Staphylococcus aureus ATCC 6538P.

c. isolation

Extract the fermentation medium twice with an equal volume of ethyl acetate, concentrate the extract to a residue in vacuo dissolve the residue in acetone and pour the acetone solution into an ethyl ether:hexane mixture (3:2) with stirring to obtain thereby a buff colored precipitate assaying about 590 μg/mg.

Alternatively, the antibiotic complex may be isolated by counter current extraction of the fermentation broth followed by concentration and precipitation as just described.

EXAMPLE 2 Separation of Antibiotic 68-1147 Components

Dissolve 1 g of antibiotic 68-1147 complex, (prepared as described in Example 1) in a mixture of acetone:benzene (3:2) and "streak out" the solution on about 25 silica gel G.F. plates 500 microns in thickness. Subject the plates to chromatography using the above-described solvent mixture. Detect the location of the components on duplicate plates by bioautography, by exposure of the plates to iodine vapors determining thereby the existence of four active components having the following Rf values:

    ______________________________________                                                68-1147 I                                                                               (0.51)                                                                68-1147 II                                                                              (0.42)                                                                68-1147 III                                                                             (0.31)                                                                68-1147 IV                                                                              (0.03)                                                         ______________________________________                                    

Isolate the respective components by scraping the appropriate band from the silica gel plate. Component I (68-1147 I) the major component weighs 320 mg and is purified by rechromatography as described above to yield 162 mg assaying 2400 μg/mg.

EXAMPLE 3 Alternate Separation of Antibiotic 68-1147 Components

Precipitate a concentrate of an ethyl acetate extract obtained from a 50 gal. fermentation in to hexane to obtain a gummy precipitate. Dissolve the gum in chloroform and wash the solution with potassium hydrogen phthalate buffer (pH 4.0) then with sodium dihydrogen phosphate buffer (pH 6.0), dry the solution over anhydrous sodium sulfate and evaporate to a residue. Crystallize the residue from acetone to obtain antibiotic 68-1147 complex, yield = 6.6 g.

Prepare a chromatographic column with 300 g. of silica gel (Type 60, Stahl). Adsorb the antibiotic complex from a mixture of acetone:benzene (4:10) and develop the column with the same solvent mixture. Monitor the chromatographic separation by thin layer chromatography using silica gel and an acetone:benzene (1:2) solvent mixture. Combine fractions having Rf values of 0.2→0.4 and evaporate to dryness-yield 2.2 g.

Rechromatograph the solids on silica gel plates 250 μ thick using acetone:benzene (5.5:10) by spotting 10 mg/plate. Monitor the separation as described above to obtain thereby 1.0 g. of 68-1147 I,Rf (0.36) M.P. 229°-231° C dec., [α]_(D) ²⁶° = -87.5° (0.4% dioxane) and (0.64 g) 68-1147 II, M.P. decomposes at about 300° C, [α]_(D) ²⁶° =-65.5° (0.4% dioxane). 

We claim:
 1. A process for producing antibiotic 68-1147 complex which comprises cultivating an antibiotic 68-1147 producing strain of Micromonospora arborensis NRRL 8041 in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen, under aerobic conditions until substantial antibiotic activity is imparted to said medium and isolating antibiotic 68-1147 complex therefrom.
 2. A process according to claim 1 wherein the microorganism is cultivated at a temperature between 20° and 40° l C for from about 2 to about 7 days and from about pH 6.0 to about pH 8.5.
 3. A process according to claim 2 wherein the microorganism is cultivated at a temperature between 28° and 35° C and from about pH 7.0 to about pH 8.0.
 4. A process according to claim 3 including the step of separating antibiotic 68-1147 I and antibiotic 68-1147II of the thus isolated antibiotic 68-1147 complex by chromatography on silica gel.
 5. Antibiotic 68-1147 I, a compound which:a. is effective in inhibiting the growth of various bacteria, especially Gram positive bacteria; b. has an optical rotation [α]_(D) ²⁶° = -87.5° (C,0.4%, dioxane) c. has a characteristic ultraviolet absorption spectrum in trifluoroethanol as follows:

    ______________________________________                                         λ max     E.sup.1%.sub.cm                                               ______________________________________                                         202 mμ        53.55                                                         247              32.55                                                         308              10.72                                                         ______________________________________                                    

d. has the following elemental analysis: C, 49.46; H, 5.20; N, 14.88; O, 16.18; S, 10.47; e. has a melting point of 229°- 231° C dec. f. has a characteristic infrared absorption spectrum in mineral oil (Nujol) with absorption at:

    ______________________________________                                         3.05 m,sp   7.30 m,sp    9.75 w,sp                                             3.45 s,sp   7.45 sh      9.95 sh                                               5.78 w,sp   7.65 w,sp   10.58 w,br                                             6.08 s      8.30 w,sp   10.70 w,br                                             6.31 w,sp   9.00 w,sp   11.20 w,br                                             6.60 s      9.15 w,br   11.30 sh                                               6.65 s      9.50 w,br   11.90 microns w,br                                     6.75 s      9.40 w,br                                                          ______________________________________                                    

g. contains the following aminoacids: threonine, proline, alanine, cysteine, isoluecine and tyrosine.
 6. Antibiotic 68-1147 II, made by the process of claim 4 being further characterized as having the following characteristics:a. is effective in inhibiting the growth of various bacteria, especially Gram positive bacteria; b. has an optical rotation [α]_(D) ²⁶° = -65.5° (C, 0.4%, dioxane); c. has a characteristic ultraviolet absorption spectrum in trifluoroethanol as follows:

    ______________________________________                                          λ max                                                                                    ##STR3##                                                     ______________________________________                                         240 mμ        18.84                                                         300 mμ        5.42                                                          ______________________________________                                    

d. has the following elemental analysis: C,50.24; H, 4.98; N, 15.87; O, 16.24; S, 12.67 (by difference); e. has a melting point ≅300° C dec., and f. contains the following amino acids: threonine, proline, alanine, isoleucine and tyrosine. 